Khadaroo Rachel G, Parodo Jean, Powers Kinga A, Papia Giuseppe, Marshall John C, Kapus Andras, Rotstein Ori D
Department of Surgery, University Health Network, and University of Toronto, Ontario, Canada.
Surgery. 2003 Aug;134(2):242-6. doi: 10.1067/msy.2003.228.
Resuscitated hemorrhagic shock predisposes patients to the development of organ dysfunction, particularly to lung injury. Ischemia/reperfusion during shock is believed to prime the immune system for an exaggerated inflammatory response to a second delayed stimulus. We previously reported an in vitro model of oxidant-induced priming of the macrophage to lipopolysaccharide (LPS) involves the Src family of tyrosine kinases. Because the Src family has been shown to activate the p38 mitogen-activated protein kinase (MAPK) pathway, we hypothesize that LPS signaling after oxidant stress involves the p38 pathway and is activated by Src kinases.
The murine macrophage cell line, Raw 264.7, was first incubated with H(2)O(2) 100 micromol/L for 1 hour and then with low dose LPS 0.01 microg/mL for 5 to 45 minutes. In a separate experiment, the cells were pretreated with PP2 or SB203580, a specific inhibitor of the Src family and p38 respectively. The phosphorylation of p38, representative of its activation, was assessed in whole cell lysates by use of Western blotting. NF-kappaB translocation was detected by immunofluorescence with anti-p65 antibody.
There is a time dependent earlier activation of p38 by oxidant stress. H(2)O(2) augmented the LPS-induced p38 phosphorylation. The Src inhibitor, PP2, prevented only the LPS-induced earlier phosphorylation after oxidant stress and had no effect on LPS activation of p38 alone. The p38 inhibitor had no effect in preventing NF-kappaB translocation in either the LPS- or H(2)O(2)/LPS-exposed cells.
Oxidant stress generated during global ischemia/reperfusion activates p38 MAPK in an Src-dependent manner. Oxidants seem to alter the LPS-induced activation of p38. P38 does not seem to have a direct role in leading to oxidant-induced NF-kappaB translocation but may affect other oxidant-induced transcription factors. This altered pathway provides an alternative avenue to target therapy during the oxidant-induced priming of the macrophage induced by trauma resuscitation.
复苏后的失血性休克使患者易发生器官功能障碍，尤其是肺损伤。休克期间的缺血/再灌注被认为会使免疫系统对第二次延迟刺激产生过度的炎症反应。我们之前报道过一种体外模型，即氧化剂诱导巨噬细胞对脂多糖（LPS）的预激活涉及酪氨酸激酶的Src家族。由于Src家族已被证明可激活p38丝裂原活化蛋白激酶（MAPK）途径，我们推测氧化应激后LPS信号传导涉及p38途径并由Src激酶激活。
将小鼠巨噬细胞系Raw 264.7首先用100微摩尔/升的H₂O₂孵育1小时，然后用0.01微克/毫升的低剂量LPS孵育5至45分钟。在另一个实验中，细胞分别用PP2或SB203580预处理，PP2是Src家族的特异性抑制剂，SB203580是p38的特异性抑制剂。通过蛋白质印迹法在全细胞裂解物中评估代表p38激活的磷酸化情况。用抗p65抗体通过免疫荧光检测NF-κB易位。
氧化应激可导致p38出现时间依赖性的早期激活。H₂O₂增强了LPS诱导的p38磷酸化。Src抑制剂PP2仅能阻止氧化应激后LPS诱导的早期磷酸化，对单独的LPS激活p38没有影响。p38抑制剂对防止LPS或H₂O₂/LPS处理的细胞中的NF-κB易位均无作用。
全身缺血/再灌注期间产生的氧化应激以Src依赖的方式激活p38 MAPK。氧化剂似乎改变了LPS诱导的p38激活。p38似乎在导致氧化应激诱导的NF-κB易位中没有直接作用，但可能影响其他氧化应激诱导的转录因子。这种改变的途径为创伤复苏诱导的巨噬细胞氧化应激预激活期间的靶向治疗提供了一条替代途径。
